Volume 63, Issue 5, Pages 827-839 (September 2016) Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase  Annika Weber, Itamar Cohen, Oliver Popp, Gunnar Dittmar, Yuval Reiss, Thomas Sommer, Tommer Ravid, Ernst Jarosch  Molecular Cell  Volume 63, Issue 5, Pages 827-839 (September 2016) DOI: 10.1016/j.molcel.2016.07.020 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Distinct Activities of Ubc6 and Ubc7 in the Ubiquitylation of Doa10 Substrates (A) Degradation of Vma12-DegAB was monitored in the given yeast strains by a cycloheximide decay assay and immunoblotting with anti-FLAG antibodies. An immunoblot using antibodies against glucose-6-phosphate dehydrogenase (G6PD) serves as loading control. WT, wild-type. The topology of Vma12-DegAB is shown in a cartoon. C′ refers to the carboxy terminus of the protein. (B–D) Vma12-DegAB (B) or Vma12-DegABDD (C and D) was isolated from lysates of the given yeast strains by FLAG affinity precipitation and analyzed by immunoblotting using anti-Ub and anti-FLAG antibodies. Where indicated, the given Ub variants were overexpressed from plasmids. Arrowheads indicate the position of mono-ubiquitylated species. The numbers on the left refer to the migration of standard proteins of known molecular weight in kilodaltons. See also Figure S1. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 In Vitro Ubiquitylation by Ubc6 and Ubc7 (A–C) Immunoblot analysis of in vitro ubiquitylation time-course experiments containing Doa10R and Ubc7/Cue1ΔTM (A), Doa10R and Ubc6ΔTM (B), or Doa10R, Ubc6ΔTM, and Ubc7/Cue1ΔTM (C). Ub blots depict overall Ub levels, containing free unanchored Ub chains as well as conjugates to Doa10R and Ubc6ΔTM. Cross-reacting signals of antibodies are indicated with asterisks. (D) In vitro ubiquitylation reactions of Doa10R, Ubc6ΔTM, and Ubc7/Cue1ΔTM with different Ub variants (Ub, UbR48, UbR63, and UbR11). A control (lane 1) was incubated with catalytically inactive Ubc6 (Ubc6S87ΔTM). Samples were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. See also Figure S2. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Mutations in Doa10R Differently Affect Ubc6 and Ubc7 Activity (A) Clustal Omega (Sievers et al., 2011) sequence alignment of the Doa10 RING domain with human and yeast APC11. Identical residues are highlighted in yellow. Residues mutated to alanine are marked with red dots. (B–D) In vitro ubiquitylation reactions containing Doa10R variants (wt Doa10R, Doa10RA41, Doa10RA43, Doa10RA73) with Ubc7/Cue1ΔTM (B), Ubc6ΔTM (C), or both E2 enzymes (D). The stimulation of Ubc7 by Doa10R variants was determined by quantifying the formation of unanchored Ub chains and normalized to the activity of Ubc7 in absence of Doa10R (B, bottom). Error bars represent SD of three independent experiments. See also Figure S3. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Ubc7 Attaches K48-pUb Directly to Mono-Ub on Doa10R (A) Doa10R was incubated with UbR48 or UbR63 in the presence of Ubc6ΔTM-His6 or Ubc6S87ΔTM-His6 as indicated. The Ubc6 variants were removed by TALON Resin, and Ub with Ubc7/Cue1ΔTM was added to the reaction. The samples were then analyzed by immunoblotting with the given antibodies. Unspecific signals due to cross-reacting antibodies are labeled with asterisks. Ub blots to determine Ubc7 activity are shown in Figure S4A. (B) Doa10R was incubated with Ubc6ΔTM in the presence of Ub, and a mono-ubiquitylated form of Doa10R was purified. Subsequently, Doa10R-Ub was supplied with UbR48 and Ubc7/Cue1ΔTM or Ubc6ΔTM, and the reaction products were analyzed by immunoblotting with the given antibodies. Of note, purified Doa10R-Ub contained minor amounts of double mono-ubiquitylated Doa10R, unmodified Doa10R, and inactivated Ubc6ΔTM. (C) Di-ubiquitylated forms of Doa10R (Doa10R-Ub2) from (B) were isolated from SDS gels, and the amount of lysine 48-linked Ub (UbK48) or total Ub in relation to standard peptides was determined by MS. Lane numbering refers to the experiment in (B). Each probe was measured three times, with two transitions monitored for each peptide. The results were averaged, and the SD of mean is shown (see also Figure S4B). (D and E) Degradation of UbV76-Ubc6S87 (Ub-Ubc6S87) (D) or UbR48,V76-Ubc6S87 (UbR48-Ubc6S87) (E) was monitored by cycloheximide decay assays in the given yeast strains. Immunoblots with antibodies against G6PD serve as loading controls. Quantification of immunoblot signals is shown in Figures S4D and S4E. The topology of un-cleavable Ub (UbV76) fused to Ubc6S87 is shown in a cartoon. C′ refers to the carboxy terminus of the protein. See also Figures S4 and S5 and Table S1. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Ubc6 Targets Amino Acids Containing Hydroxyl Groups (A) In vitro ubiquitylation of Ubc6 variants (wt Ubc6ΔTM, Ubc6A196ΔTM) with fluorescently labeled ubiquitin. Where indicated, samples were treated with 100 mM NaOH to hydrolyze ester linkages (lanes 4, 6, and 8). Reactions lacking ATP serve as controls (lanes 1 and 2). Samples were analyzed by immunoblotting or fluorescence scanning (Ub-Alexa488). (B) Mass spectrometric analysis of the serine ubiquitylation site on Ubc6ΔTM after incubation with Ub, E1, and ATP. Ubc6-Ub was isolated from Coomassie-stained gels and processed as described in Experimental Procedures. The b- and y-fragmentation pattern of the tryptic peptide, spanning residues 194–206, shows the formation of an ester bond between the serine side chain and the C terminus of ubiquitin. The ubiquitylated peptide was recorded at 949.99 m/z with a pep score of 254.72. Identified fragment ions are shown in the fragmentation legend. The modified serine 196 is included in both the b- and the y-series of fragment ions. (C) Schematic drawing of the chemical structure of the ubiquitin-ester bond to the serine side chain at position 196 of Ubc6. (D) Cell lysate was prepared from strains expressing either wt Ubc6 or Ubc6A196, incubated with 150 mM NaOH, where indicated, and analyzed by immunoblotting with antibodies against Ubc6 to detect mono-ubiquitylated species. See also Figure S6. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Ubc6 Conjugation Sites Are Spatially Flexible (A) Sequence of Sbh2 showing the position of lysine residues and the transmembrane domain. Lysine residues that were mutated to arginine residues (Sbh2Δ4K) are marked with red dots. The topology of Sbh2 is presented in a cartoon. C′ refers to the carboxy terminus of the protein. (B and C) Quantification of cycloheximide decay assays to monitor the turnover of Sbh2 (B) and Sbh2Δ4K (C) in cells deleted for SSH1 (ssh1Δ). The error bars represent the SD of three independent experiments. Representative immunoblots are shown in Figure S7A. (D) Degradation of FLAG-tagged Sbh2 or Sbh2Δ4K and their Ub fusions was monitored by cycloheximide decay assays in ubc6Δ yeast cells. Immunoblots with antibodies against G6PD serve as loading controls. Quantification of immunoblot signals is given in Figure S7B. The topology of un-cleavable Ub (UbV76) fused to Sbh2 is shown in a cartoon. C′ refers to the carboxy terminus of the protein. (E) Immunoprecipitation of FLAG-Sbh2 and FLAG-Sbh2Δ4K from lysates derived of Δssh1 rpt4R yeast cells harboring deletions of the indicated genes. In all cells, Myc-Ub was overexpressed from a plasmid. Immunoblots showing the input material are given in Figure S7C. (F) Immunoprecipitation of FLAG-Sbh2 and FLAG-Sbh2Δ4K from ssh1Δ yeast cells overexpressing Myc-Ub. Samples were split to two, and, where indicated, NaOH was added to the sample buffer prior to loading on gels (lanes 4–6). Control precipitations from cell extracts that do not contain FLAG-tagged proteins are shown in lanes 1 and 4. Immunoblots showing the input material are given in Figure S7D. See also Figure S7. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 A Model of the Tandem Activity of Ubc6 and Ubc7 at the Doa10 Ligase (A) In the initial step, Ubc6 attaches short Ub conjugates to lysine residues (K) but also to hydroxylated amino acids such as serine (S) or threonine (T) of Doa10 substrates (priming). (B) These primary Ub molecules label polypeptides for subsequent conjugation of K48-pUb chains by Ubc7 (elongation). (C) In some cases, when lysine residues are presented in an appropriate context, Ubc7 can directly add Ub to client molecules, which partly supersedes Ubc6 activity. Molecular Cell 2016 63, 827-839DOI: (10.1016/j.molcel.2016.07.020) Copyright © 2016 Elsevier Inc. Terms and Conditions